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References and Notes 1. J. C. Farman, B. G. Gardiner, J. D. Shanklin, Nature 315, 207 (1985). 2. World Meteorological Organization (WMO), Scientific Assessment of Ozone Depletion: 2006 (WMO, Geneva, Switzerland, 2007); http://ozone.unep.org/ Assessment_Panels/SAP/Scientific_Assessment_2006.
3. F. D. Pope, J. C. Hansen, K. D. Bayes, R. R. Friedl, S. P. Sander, J. Phys. Chem. A 111, 4322 (2007). 4. Q. Schiermeier, Nature 449, 382 (2007). 5. M. von Hobe et al., Atmos. Chem. Phys. 7, 3055 (2007). 6. M. von Hobe, Science 318, 1878 (2007). 7. S. P. Sander et al., Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies 06-2 ( Jet Propulsion Laboratory, Pasadena, CA, 2006). 8. R. A. Cox, G. D. Hayman, Nature 332, 796 (1988). 9. J. B. Burkholder, J. J. Orlando, C. J. Howard, J. Phys. Chem. 94, 687 (1990). 10. K. J. Huder, W. B. DeMore, J. Phys. Chem. 99, 3905 (1995). 11. W. B. DeMore, E. Tschuikow-Roux, J. Phys. Chem. 94, 5856 (1990). 12. L. T. Molina, M. J. Molina, J. Phys. Chem. 91, 433 (1987). 13. W. J. Bloss, S. L. Nickolaisen, R. J. Salawitch, R. R. Friedl, S. P. Sander, J. Phys. Chem. A 105, 11226 (2001). 14. J. R. McKeachie, M. F. Appel, U. Kirchner, R. N. Schindler, Th. Benter, J. Phys. Chem. B 108, 16786 (2004). 15. J. Plenge et al., J. Phys. Chem. A 109, 6730 (2005). 16. T. Ingham, S. P. Sander, R. R. Friedl, Faraday Discuss. 130, 89 (2005). 17. R. Broske, F. Zabel, J. Phys. Chem. A 110, 3280 (2006). 18. J. J. Lin, D. W. Hwang, S. Harich, Y. T. Lee, X. Yang, Rev. Sci. Instrum. 69, 1642 (1998). 19. M. von Hobe, F. Stroh, H. Beckers, T. Benter, H. Willner, Phys. Chem. Chem. Phys. 11, 1571 (2009). 20. C. L. Lin, J. Chem. Eng. Data 21, 411 (1976). 21. A. L. Kaledin, K. Morokuma, J. Chem. Phys. 113, 5750 (2000).
Host Inhibition of a Bacterial Virulence Effector Triggers Immunity to Infection Vardis Ntoukakis, Tatiana S. Mucyn, Selena Gimenez-Ibanez, Helen C. Chapman, Jose R. Gutierrez, Alexi L. Balmuth, Alexandra M. E. Jones, John P. Rathjen* Plant pathogenic bacteria secrete effector proteins that attack the host signaling machinery to suppress immunity. Effectors can be recognized by hosts leading to immunity. One such effector is AvrPtoB of Pseudomonas syringae, which degrades host protein kinases, such as tomato Fen, through an E3 ligase domain. Pto kinase, which is highly related to Fen, recognizes AvrPtoB in conjunction with the resistance protein Prf. Here we show that Pto is resistant to AvrPtoB-mediated degradation because it inactivates the E3 ligase domain. AvrPtoB ubiquitinated Fen within the catalytic cleft, leading to its breakdown and loss of the associated Prf protein. Pto avoids this by phosphorylating and inactivating the AvrPtoB E3 domain. Thus, inactivation of a pathogen virulence molecule is one mechanism by which plants resist disease. he effector proteins of bacterial, fungal, and oomycete plant pathogens collectively determine pathogenicity on susceptible host species. However, resistance (R) proteins of the plant immune system can recognize these effectors and restrict pathogen growth. The largest class of R proteins is the nucleotide-binding site– plus–leucine-rich repeat (NB-LRR) class (1). Recognition of pathogen effectors by R proteins not only protects crops from pathogen attack, but also controls important immune responses in animals (2). Despite their importance, R proteins are poorly understood at a mechanistic level. Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a pathogen of tomato and Arabidopsis, and it in-
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The Sainsbury Laboratory, Colney, Norwich NR4 7UH, UK. *To whom correspondence should be addressed. E-mail:
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jects ~30 effectors into host cells (3). One of these effectors, AvrPtoB, degrades host protein kinases through ubiquitination and proteasomal degradation mediated by a C-terminal E3 ubiquitin ligase domain (4–6). AvrPtoB is widely conserved among diverse bacterial pathogens, including Xanthomonas, Erwinia, and many strains of Pseudomonas (7). One target of AvrPtoB is the tomato kinase Fen (4), which signals together with the NB-LRR resistance protein Prf. AvrPtoB mutants lacking ubiquitin ligase activity elicit a host immune response mediated by Fen/Prf, leading to defense gene induction and localized cell death known as the hypersensitive response. In resistant cultivars, AvrPtoB is recognized by a protein complex composed of Prf and the Pto kinase, which is highly related (80% identity) to Fen (8–10). The inability of AvrPtoB to degrade Pto underlies its recognition, but its molecular mechanism is not yet understood.
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22. A. Toniolo, G. Granucci, S. Inglese, M. Persico, Phys. Chem. Chem. Phys. 3, 4266 (2001). 23. K. A. Peterson, J. S. Francisco, J. Chem. Phys. 121, 2611 (2004). 24. T. A. Moore, M. Okumura, J. W. Seale, T. K. Minton, J. Phys. Chem. A 103, 1691 (1999). 25. R. Aures et al., J. Chem. Phys. 117, 2141 (2002). 26. T. A. Moore, M. Okumura, T. K. Minton, J. Chem. Phys. 107, 3337 (1997). 27. S. L. Nickolaisen et al., J. Chem. Phys. 104, 2857 (1996). 28. Materials and methods are available as supporting material on Science Online. 29. M. Birk et al., J. Chem. Phys. 91, 6588 (1989). 30. R. M. Stimpfle, D. M. Wilmouth, R. J. Salawitch, J. G. Anderson, J. Geophys. Res. 109, D03301 (2004). 31. This work is supported by Academia Sinica and National Science Council (NSC 95-2113-M-001-041-MY3), Taiwan. We thank M.-C. Liang for bringing our attention to the ClOOCl issue and K. A. Boering and Y.-P. Lee for valuable comments.
Supporting Online Material www.sciencemag.org/cgi/content/full/324/5928/781/DC1 Materials and Methods Figs. S1 to S3 Table S1 References 23 January 2009; accepted 17 March 2009 10.1126/science.1171305
One important difference between Pto and Fen is their relative kinase activities. Pto autophosphorylation activity in vitro was substantially higher than that of Fen (Fig. 1A), and Pto (but not Fen) was phosphorylated in vivo after transient expression in Nicotiana benthamiana leaves (fig. S1) (11). Fen was substantially less active than Pto in phosphorylating the in vitro substrate Pti1 (12) (fig. S2). While studying interactions between effector protein AvrPtoB and the host kinases, we found that both Pto and Fen were able to phosphorylate AvrPtoB (Fig. 1A). Again Pto was more active, with a Michaelis-Menton constant (Km) of 2.2 mM compared with 10 mM for Fen. A kinase mutant, PtoD164N (13), did not autophosphorylate or transphosphorylate AvrPtoB in these assays. We mapped the phosphorylation site on AvrPtoB to Thr450 using mass spectrometry analysis, based on the series of y and b ions (Fig. 1B and fig. S3). This residue lies in the E3 ligase domain and is conserved among AvrPtoB homologs from different P. syringae strains (14). Protein phosphorylation is important in the regulation of E3 ligases (15–18). To test the effect of phosphorylation on AvrPtoB E3 ligase activity, we analyzed autoubiquitination in the presence of Pto with the use of recombinant proteins. Increasing amounts of Pto decreased the levels of polyubiquitinated AvrPtoB (Fig. 2A). Similarly, Pto inhibited the trans-ubiquitination of Fen by AvrPtoB in vitro, in a dose-dependent manner (Fig. 2B). Conversely, Fen inhibited its own ubiquitination only when it was allowed to prephosphorylate AvrPtoB (fig. S4), consistent with its weaker kinase activity. Thus, the in vitro data suggest that ubiquitination and phosphorylation are competitive processes that determine the outcome of the AvrPtoB-Pto interaction. Substitution
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cycle is an even more efficient process for polar ozone loss than previously thought (2). As for the Pope 2007 cross sections (3), it is likely that the authors’ functional fitting method may overcorrect for the Cl2 absorbance, resulting in cross sections that are too small in the wavelength region at which Cl2 absorbs significantly (>300 nm). Although at this point we can only report the cross sections of ClOOCl at two wavelengths because of our requirement of high-intensity lasers, these measurements are surely sufficient to resolve the discrepancies in the photolysis rates of ClOOCl and to restore confidence in standard photochemical models for ozone degradation. We have also demonstrated a promising method for measuring reliable laboratory cross sections of important species in atmospheric chemistry, free from interference by impurities.
REPORTS mutants of Thr450 could not be phosphorylated by Pto or Fen (fig. S5) and displayed no E3 ligase activity in vitro (Fig. 2C and fig. S6). The phosphomimetic mutant, AvrPtoBT450D, caused cell death upon transient expression in N. benthamiana leaves, as described for other AvrPtoB E3 ligase mutants (19) (fig. S7). We next investigated whether AvrPtoBT450D was able to trigger immunity on susceptible tomatoes, like other E3 ligase mutants such as AvrPtoBF479A (4). In these experiments, we used the Rio Grande tomato line, which is resistant to Pst DC3000 infection, containing both Pto and Prf genes (RG-pto11) and two isogenic susceptible lines containing the prf-3 (RG-prf3) or pto-11 (RG pto-11) mutations (20). All lines carried the Fen gene. These plants were infected with a Pst DC3000 strain containing deletions in the avrPto and avrPtoB genes (21) expressing an empty vector control, AvrPtoB, or the E3 ligase mutants AvrPtoBT450D and AvrPtoBF479A (Fig. 2D). Whereas all bacterial strains grew to similar levels on the line lacking Prf, only the vector control grew on the resistant line, indicating that the Pto/Prf complex recognized AvrPtoB and its mutant forms. Growth of bacteria expressing AvrPtoBT450D or AvrPtoBF479A, but not wild-type (WT) AvrPtoB, was severely restricted on the line containing the pto-11 mutation, indicating that the mutants were recognized by Fen (4). Thus, the host kinase Pto probably
Fig. 1. Pto phosphorylates AvrPtoB within the E3 ligase domain. (A) Kinase activity assay showing Pto and Fen autophosphorylation and transphosphorylation of AvrPtoB. The assays were conducted as described previously (26) on purified proteins, as indicated. Proteins were fractionated on SDS– polyacrylamide gel electrophoresis before transfer to polyvinylidene difluoride membranes for autoradiography. (B). Liquid chromatography–mass spectrometry analysis of phosphorylated AvrPtoB identified peptide A436ALDPIASQFSQLRTISK. The fragmentation pattern is presented, possible cleavages are indicated by vertical bars, and observed b and y ions are numbered. Full spectra are presented in fig. S3.
abrogates the ubiquitination activity of AvrPtoB by phosphorylating it within the E3 ligase domain. To further investigate this, we mapped AvrPtoBdependent ubiquitination sites on Fen generated in vitro. We identified one ubiquitinated peptide, DVK164CTNILLDENFVPK (22) (fig. S8). A Lys164 substitution mutant, FenK164R, was not polyubiquitinated by AvrPtoB, either in vitro or in vivo, in the presence of the proteasome inhibitor MG132 (Fig. 3, A and B). This residue is invariant in protein kinases and contributes to the mechanism of catalytic phosphotransfer (23); thus, AvrPtoB may target this residue generally to inactivate host protein kinases. The greater kinase activity of Pto relative to Fen may explain its ability to resist ubiquitination by AvrPtoB. To test this possibility, we examined the ubiquitination of the kinase mutant PtoD164N. Whereas both Fen (4) and PtoD164N were polyubiquitinated by AvrPtoB in vitro, WT Pto was not (Fig. 3A). Similarly in N. benthamiana, Fen and PtoD164N, but not WT Pto, were polyubiquitinated by AvrPtoB in the presence of MG132 (Fig. 3B) and degraded in its absence (fig. S9). These data
suggest a mechanism in which Fen is polyubiquitinated by AvrPtoB within the catalytic cleft of the kinase, leading to its proteasomal degradation, whereas Pto avoids this by phosphorylating and inhibiting the E3 ligase domain of AvrPtoB. Pto resides in a constitutive molecular complex with the resistance protein Prf (9), and Prf was unstable in the absence of Pto homologs because of proteasomal degradation (fig. S10). We tested to determine whether Fen degradation induced by AvrPtoB diminished Prf levels. Resistant tomatoes expressing Pto, Fen, and Prf or an isogenic susceptible line containing the pto-11 mutation were infected with Pst DC3000 or a deletion mutant lacking the avrPtoB gene (21). No loss of Prf was visible in the tomato line containing Pto. In contrast, there was a severe decrease of Prf levels in the line lacking Pto, in the presence but not absence of AvrPtoB (Fig. 4A). To further test if AvrPtoB degrades Prf in a proteasome-dependent manner, we used stable transgenic N. benthamiana lines expressing the tomato Prf and/or Pto genes (24). Transient expression of AvrPtoB resulted in Prf degradation,
Fig. 2. Phosphorylation of AvrPtoB by Pto inhibits its E3 ligase activity. (A) Effector protein AvrPtoB autoubiquitination assay with increasing amounts of host kinase Pto, analyzed by immunoblot. (B) Ubiquitination assay of Fen kinase by AvrPtoB with increasing amounts of Pto, analyzed by immunoblot. (C) In vitro ubiquitination assay consisting of ubiquitination reaction mixture with FLAG-Ub, containing glutathione S-transferase (GST)–AvrPtoB or GST-AvrPtoBT450D as indicated. Proteins were analyzed by immunoblot. (D) Bacterial growth on tomato leaves infected with a Pst DC3000 strain containing deletions in the avrPto and avrPtoB genes, expressing an empty vector control, AvrPtoB, the phosphomimetic mutant AvrPtoBT450D, or the E2-binding mutant AvrPtoBF479A. The bars represent bacterial growth after three days of infection. At 0 days, the bacterial leaf titer was uniformly ~3.0 log [colonyforming units (cfu) cm–2] for all treatments. Data represent two independent replicate experiments and error bars T SE (n = 4 samples).
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REPORTS dependent on proteasome function and the E3 ligase activity of AvrPtoB (Fig. 4B). Similar to the tomato experiments, Pto protected Prf from AvrPtoB-mediated degradation. Both Prf and PtoD164N were degraded in the presence of AvrPtoB (fig. S11). Thus, Prf loss was attributable to instability of the associated kinase, and as such, Prf is an indirect target of the effector protein. We have not found evidence of direct interaction between Prf and AvrPtoB. The data suggest an indirect mechanism of degradation where the fate of the Prf complex depends on the activity of the specific kinase within the complex. The results above show a clear role for Pto kinase activity in AvrPtoB inactivation. Signaling by Pto is believed to be kinase-independent downstream of
Pto activation (13, 25). To test a role for Pto kinase activity beyond phosphorylation of AvrPtoB, we examined elicitation of immunity by an AvrPtoB E3 ligase mutant. In these experiments, AvrPtoBF479A was delivered by P. syringae pv. tabaci or Agrobacterium on transgenic N. benthamiana plants expressing both PtoD164N and Prf. The Pto kinase mutant was able to recognize the AvrPtoB E3 ligase mutant, leading to functional disease resistance and induction of cell death (Fig. 4C and fig. S12). Thus, in these experiments, Pto kinase activity was important only for inactivation of AvrPtoB E3 ligase activity. Plant immunity against bacteria relies on recognition of bacterial effectors by R proteins. In many cases, R proteins are organized into protein
complexes capable of recognizing the effectors and activating signal transduction (1). Our results demonstrate a mechanism in which the bacteria inject an effector into the plant cell to target the stability of a resistance complex, among other known targets (5, 6). The outcome of the interaction depends on competition between the phosphorylation and ubiquitination events mediated by the host kinase and bacterial effector, respectively. Pto represents a host variant molecule with higher kinase activity and, hence, the ability to fight back against the effector. Phosphorylation of AvrPtoB restores the normal function of effector recognition. Overall, this study describes host enzymatic inactivation of a bacterial virulence effector to confer immunity.
Fig. 3. Ubiquitination of Fen and Pto by AvrPtoB. (A) In vitro assays performed with ubiquitination reaction mixture and the indicated His-tagged fusions proteins. Pto and Fen were detected as polyubiquitinated [Pto/Fen (Ub)n], oligoubiquitinated, or unmodified proteins, with an a-His antibody. PtoD164N is a kinase-deficient mutant; FenK164R contains a substitution in an in vitro ubiquitinated residue. Polyubiquitinated forms of AvrPtoB [AvrPtoB (Ub)n] were detected with a-AvrPtoB antisera. (B) Ubiquitination of Fen and PtoD164N by AvrPtoB in vivo. N. benthamiana leaves were transiently transformed with constructs expressing the proteins as indicated, in the presence of the proteasome inhibitor MG132, and the results were analyzed by immunoblot. Pto and Fen were detected in crude extracts and after immunoprecipitation as unmodified or polyubiquitinated [Pto/Fen (Ub)n] proteins, with the use of an a-FLAG antibody.
Fig. 4. AvrPtoB targets the Prf signaling complex for degradation. (A) Immunoblot of Prf levels in tomato leaves containing Pto (RG-PtoR) or lacking Pto (RG-pto11) after bacterial infection with Pst DC3000 or a deletion mutant Pst DC3000DavrPtoB lacking the AvrPtoB gene. Coomassie brilliant blue (CBB) staining of the membrane confirmed equal gel loading. (B) Prf degradation by AvrPtoB is proteasome-mediated and suppressed by the kinase activity of Pto. Stable transgenic N. benthamiana lines expressing tomato Prf only, Prf and Pto, or Prf and PtoD164N genes were transiently transformed with 35S:AvrPtoB or 35S:AvrPtoBF479A as indicated, in the presence or
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absence of the proteasome inhibitor MG132, and they were analyzed by immunoblot. CBB staining of the membrane confirmed equal gel loading. (C) Bar chart of bacterial growth of P. syringae pv. tabaci (Pta) 11528 bacteria expressing either empty vector (EV), AvrPtoB, or AvrPtoBF479A after 6 days of infection on the stable transgenic lines from (B). At 0 days, the bacterial leaf titer was uniformly ~1.0 log (cfu cm–2) for all treatments. Data represent two independent replicate experiments. Asterisks indicate significant (P < 0.01) reduction in bacterial growth and error bars T SE (n = 4).
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REPORTS References and Notes 1. J. L. Dangl, J. D. G. Jones, Nature 411, 826 (2001). 2. W. Strober, P. J. Murray, A. Kitani, T. Watanabe, Nat. Rev. Immunol. 6, 9 (2006). 3. L. M. Schechter et al., Mol. Plant-Microbe Interact. 19, 1180 (2006). 4. T. R. Rosebrock et al., Nature 448, 370 (2007). 5. V. Gohre et al., Curr. Biol. 18, 1824 (2008). 6. S. Gimenez-Ibanez et al., Curr. Biol. 19, 423 (2009). 7. R. B. Abramovitch, Y.-J. Kim, S. Chen, M. B. Dickman, G. B. Martin, EMBO J. 22, 60 (2003). 8. R. B. Abramovitch, G. B. Martin, FEMS Microbiol. Lett. 245, 1 (2005). 9. T. S. Mucyn et al., Plant Cell 18, 2792 (2006). 10. R. Janjusevic, R. B. Abramovitch, G. B. Martin, C. E. Stebbins, Science 311, 222 (2006); 21 December 2005 (10.1126/ science.1120131). 11. Materials and methods are available as supporting material on Science Online. 12. J. Zhou, Y.-T. Loh, R. A. Bressan, G. B. Martin, Cell 83, 925 (1995).
13. A.-J. Wu, V. M. E. Andriotis, M. C. Durrant, J. P. Rathjen, Plant Cell 16, 2809 (2004). 14. N. C. Lin, R. B. Abramovitch, Y. J. Kim, G. B. Martin, Appl. Environ. Microbiol. 72, 702 (2006). 15. R. Kulikov, K. A. Boehme, C. Blattner, Mol. Cell. Biol. 25, 7170 (2005). 16. C. Yang et al., Mol. Cell 21, 135 (2006). 17. M. Gao et al., Science 306, 271 (2004); published online 9 September 2004 (10.1126/science.1099414). 18. K. Tatematsu, N. Yoshimoto, T. Okajima, K. Tanizawa, S. Kuroda, J. Biol. Chem. 283, 11575 (2008). 19. R. B. Abramovitch, R. Janjusevic, C. E. Stebbins, G. B. Martin, Proc. Natl. Acad. Sci. U.S.A. 103, 2851 (2006). 20. J. M. Salmeron, S. J. Barker, F. M. Carland, A. Y. Mehta, B. J. Staskawicz, Plant Cell 6, 511 (1994). 21. N. C. Lin, G. B. Martin, Mol. Plant-Microbe Interact. 18, 43 (2005). 22. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer Chris Tran,1* Samedy Ouk,5* Nicola J. Clegg,1 Yu Chen,1,3 Philip A. Watson,1 Vivek Arora,1 John Wongvipat,1 Peter M. Smith-Jones,2 Dongwon Yoo,5 Andrew Kwon,1 Teresa Wasielewska,1 Derek Welsbie,6 Charlie Degui Chen,6† Celestia S. Higano,7 Tomasz M. Beer,8 David T. Hung,9 Howard I. Scher,3 Michael E. Jung,5‡ Charles L. Sawyers1,4‡ Metastatic prostate cancer is treated with drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease called castration-resistant prostate cancer, driven by elevated expression of the androgen receptor. Here we characterize the diarylthiohydantoins RD162 and MDV3100, two compounds optimized from a screen for nonsteroidal antiandrogens that retain activity in the setting of increased androgen receptor expression. Both compounds bind to the androgen receptor with greater relative affinity than the clinically used antiandrogen bicalutamide, reduce the efficiency of its nuclear translocation, and impair both DNA binding to androgen response elements and recruitment of coactivators. RD162 and MDV3100 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer. reatment of advanced prostate cancer is limited by the development of resistance to antiandrogen therapy. Castration-resistant prostate cancer (CRPC) is commonly associated with increased androgen receptor (AR) gene expression, which can occur through AR gene amplification or other mechanisms (1, 2). Elevated AR expression is necessary and sufficient to confer resistance to antiandrogen therapy in mouse xenograft models (3). In addition, firstgeneration AR antagonists such as bicalutamide (also called Casodex) or flutamide demonstrate agonist properties in cells engineered to express higher AR amounts. The partial agonism of these compounds is a potential liability, best illustrated clinically by the antiandrogen withdrawal response in which serum concentrations of prostatespecific antigen (PSA) decline in patients after
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discontinuation of either of these AR antagonists (4). Collectively, these findings implicate increased AR expression as a molecular cause of drug resistance and suggest that secondgeneration antiandrogens might be identified by their ability to retain antagonism in cells expressing excess AR. Our earlier mutagenesis studies revealed that increased AR expression conferred resistance to antiandrogens in model systems only when the receptor contains a functional ligand binding domain (LBD) (3). Second-generation antiandrogens could, in theory, be optimized to exploit this well-characterized LBD. Co-crystal structures of wild-type AR bound to antagonists have not been solved, but a co-crystal of bicalutamide with mutant AR (in an agonist conformation), together with structural knowl-
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23. 24. 25. 26.
M. Huse, J. Kuriyan, Cell 109, 275 (2002). A. Balmuth, J. P. Rathjen, Plant J. 51, 978 (2007). W. Xing et al., Nature 449, 243 (2007). V. M. Andriotis, J. P. Rathjen, J. Biol. Chem. 281, 26578 (2006). 27. We thank L. Serazetdinova for help with mass spectroscopy and G. Martin (Cornell University) for bacterial strains and RG-pto11 mutant seeds. Part of this work was funded by a Biotechnology and Biological Science Research Council grant BB/D00456X/1 to J.P.R. We gratefully acknowledge the support of the Gatsby Charitable foundation.
Supporting Online Material www.sciencemag.org/cgi/content/full/324/5928/784/DC1 Materials and Methods Figs. S1 to S12 References 5 December 2008; accepted 24 February 2009 10.1126/science.1169430
edge of estrogen receptor–a (ER-a) antagonists (5), suggests a steric clash mechanism in which the bulky phenyl ring on bicalutamide leads to a partial unfolding of AR (6). However, bicalutamide has relatively low affinity for AR [at least 30-fold reduced relative to the natural ligand dihydrotestosterone (DHT)] (7), suggesting that antagonism could be optimized by improved binding characteristics. To search for improved antiandrogens, we selected the nonsteroidal agonist RU59063 as a starting chemical scaffold on the basis of its relatively high affinity for AR (only threefold reduced compared to testosterone) and selectivity for AR over other nuclear hormone receptors (8, 9). Through an iterative process to be described in detail separately (see also U.S. Patent Application 20070004753), we evaluated nearly 200 thiohydantoin derivatives of RU59063 for AR agonism and antagonism in human prostate cancer cells engineered to express increased amounts of AR. On the basis of these structure-activity relationships and further chemical modifications to improve serum 1 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 2 Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 3Genitourinary Oncology Service, Division of Solid Tumor Oncology and Sidney Kimmel Center for Prostate and Urologic Cancers, Memorial SloanKettering Cancer Center, New York, NY 10065, USA. 4Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 5Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA. 6Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA. 7 Division of Oncology, Departments of Medicine and Urology, University of Washington, Fred Hutchinson Cancer Center, Seattle, WA 98109, USA. 8OHSU Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA. 9Medivation, Inc., 201 Spear Street, San Francisco, CA 94105, USA.
*These authors contributed equally to this work. †Present address: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. ‡To whom correspondence should be addressed. E-mail:
[email protected];
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Host Inhibition of a Bacterial Virulence Effector Triggers Immunity to Infection Vardis Ntoukakis et al. Science 324, 784 (2009); DOI: 10.1126/science.1169430
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